Do longer sequences improve the accuracy of identification of forensically important Calliphoridae species? Sara Bortolini1, Giorgia Giordani2, Fabiola Tuccia2, Lara Maistrello1 and Stefano Vanin2 1 Department of Life Sciences, University of Modena and Reggio Emilia, Reggio Emilia, Italy 2 School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom ABSTRACT Species identification is a crucial step in forensic entomology. In several cases the calculation of the larval age allows the estimation of the minimum Post-Mortem Interval (mPMI). A correct identification of the species is the first step for a correct mPMI estimation. To overcome the difficulties due to the morphological identification especially of the immature stages, a molecular approach can be applied. However, difficulties in separation of closely related species are still an unsolved problem. Sequences of 4 different genes (COI, ND5, EF-1α, PER) of 13 different fly species collected during forensic experiments (Calliphora vicina, Calliphora vomitoria, Lu- cilia sericata, Lucilia illustris, Lucilia caesar, Chrysomya albiceps, Phormia regina, Cyno- mya mortuorum, Sarcophaga sp., Hydrotaea sp., Fannia scalaris, Piophila sp., Megaselia scalaris) were evaluated for their capability to identify correctly the species. Three concatenated sequences were obtained combining the four genes in order to verify if longer sequences increase the probability of a correct identification. The obtained results showed that this rule does not work for the species L. caesar and L. illustris. Future works on other DNA regions are suggested to solve this taxonomic issue. Subjects Entomology, Taxonomy Submitted 19 March 2018 Keywords ND5, COI, PER, Diptera, EF-1α, Maximum-likelihood, Phylogeny Accepted 17 October 2018 Published 17 December 2018 Corresponding author INTRODUCTION Stefano Vanin, [email protected] Species identification is a crucial step in forensic entomology. In particular, the calculation Academic editor of the age of the insects collected from a cadaver or a crime scene allows the estimation of Ilaria Negri the time of oviposition that, except in case of myiasis, can be considered as the minimum Additional Information and Post-Mortem Interval (mPMI) (Erzinclioglu,ˆ 1983; Marchenko, 1982; Smith, 1986; Amendt Declarations can be found on et al., 2007; Vanin et al., 2017). This method is particularly precise when insects of the page 13 first colonization wave—mainly Diptera in the family Calliphoridae, Sarcophagidae DOI 10.7717/peerj.5962 and Muscidae—are considered. Insect development is temperature dependent and each Copyright species has a different growth rate. Thus, the correct identification of the species leads to an 2018 Bortolini et al. accurate mPMI estimation. Species identification is classically performed by morphological Distributed under analysis of the specimens, but the lack of complete identification keys for immature stages Creative Commons CC-BY 4.0 represents a limitation to this approach. In the last twenty years, to overcome this limit, OPEN ACCESS several authors have suggested a DNA-based identification method which is frequently How to cite this article Bortolini S, Giordani G, Tuccia F, Maistrello L, Vanin S. 2018. Do longer sequences improve the accuracy of identification of forensically important Calliphoridae species? PeerJ 6:e5962 http://doi.org/10.7717/peerj.5962 used today because of the new sequencing technologies and the relative reduction of the costs (Benecke, 1998; Sperling, Anderson & Hickey, 1994; Stevens & Wall, 1996). Most of the publications about identification of forensically important species focused on the analysis of the genes coding for the cytochrome c oxidase subunit I (COI) as summarized by Tuccia and co-workers (Tuccia, Giordani & Vanin, 2016) and cytochrome c oxidase subunit II (COII) (Sperling, Anderson & Hickey, 1994; Aly, Wen & Wang, 2013; Boehme, Amendt & Zehner, 2012; Malgorn & Coquoz, 1999; Wallman & Donnellan, 2001; Xiong et al., 2012). Tested target markers other than COI and COII were: Cytb (GilArriortua et al., 2013; GilArriortua et al., 2014; GilArriortua et al., 2015; Giraldo, Uribe & Lopez, 2011), ND1 (Giraldo, Uribe & Lopez, 2011), ND5 (Zaidi et al., 2011; Zehner et al., 2004), 28S rDNA (Gibson et al., 2011; McDonagh & Stevens, 2011; Stevens & Wall, 2001; Tourle, Downie & Villet, 2009), mt16S rDNA (Guo et al., 2014; Li et al., 2010), CAD (Gibson et al., 2011; Meiklejohn et al., 2013; Schnell Schühli, Barros de Carvalho & Wiegmann, 2007), EF-1α (Gibson et al., 2011; Schnell Schühli, Barros de Carvalho & Wiegmann, 2007; McDonagh, García & Stevens, 2009), ITS1 (Zaidi et al., 2011), ITS2 (GilArriortua et al., 2014; GilArriortua et al., 2015; Zaidi et al., 2011; Ferreira et al., 2011; Nelson, Wallman & Dowton, 2008; Song, Wang & Liang, 2008; Yusseff-Vanegas & Agnarsson, 2017), PER (Guo et al., 2014) and Bicoid (Park et al., 2013). Analysis of mitochondrial DNA (mtDNA), in particular COI gene, seems to provide good species identification among Diptera, although a correct identification is still problematic for closely related species (Tourle, Downie & Villet, 2009; Harvey et al., 2008; Sonet et al., 2012). Nuclear DNA, especially ITS2 gene, presents a lack of intra-specific genetic divergence but high inter-specific variation in the genus Lucilia Robineau-Desvoidy, 1830, leading to a better resolution of closely related species (GilArriortua et al., 2014; GilArriortua et al., 2015). ITS2 was able to fully resolve the relationship within the species in the genus Cochliomyia Townsend 1915 (Yusseff-Vanegas & Agnarsson, 2016), otherwise, the same gene appeared to be not useful for Chrysomya Robineau-Desvoidy, 1830 genus studies (Nelson, Wallman & Dowton, 2008). Previous works indicate that the combination of nuclear and mitochondrial markers is a much more accurate approach for species identification. In a recent paper, the study of Caribbean blow-flies through DNA markers highlights the possibility to resolve phylogenetic relations using a combination of COI and ITS2 genes. In fact, COI failed in demonstrating a monophyly in recently diverged species, leading to uncertain identification. The addition of a second nuclear marker, such as ITS2, increases certainty in species identification (Yusseff-Vanegas & Agnarsson, 2017). McDonagh and co-worker tested a multi- loci approach (28S rRNA, COI and EF-1α) finding that multiple-gene phylogenies permit the use of genes that have evolved at different rates, and also allow the identification of experimental errors in species identification and sequencing (McDonagh & Stevens, 2011). Zaidi et al. (2011) based the identification of Diptera species on five genes and demonstrated that such a multi-gene approach allows to overcome and clarify the misdiagnosis given by a single gene identification. We focused our attention on dipteran specimens morphologically identified in order to evaluate the accuracy of the molecular approach in the identification of forensically important species. Sequences of four different markers, two mitochondrial (COI and Bortolini et al. (2018), PeerJ, DOI 10.7717/peerj.5962 2/18 ND5) and two nuclear (EF-1α and PER) were used. According to literature, identification based on a single gene had showed discordant outcomes compared with morphological results (Meier et al., 2006; Vilgalys, 2003) especially in the case of closely related species. In order to clarify the accuracy of a molecular multiple-loci approach in the identification of forensically important species, we built concatenated sequences using the four different markers. MATERIALS AND METHODS Eighty specimens (Table 1) were collected between 2011 and 2014 in Italy (Emilia Romagna, Veneto and Calabria), England (West Yorkshire) and Belgium, and preserved in absolute ethanol. The specimens were observed under the microscope and identified using taxonomic keys (Table 2). DNA extraction from adult insects was performed on abdominal tissues carefully dissected, to prevent external contaminations and to preserve the external structure of the insect for future examination. Full puparia and larvae were instead entirely processed, after a photographic documentation to allow further observations. DNA was extracted using the QIAamp DNA Mini Kit R (QIAGEN, Germantown, MD, USA), following the manufacture protocol ``DNA Purification from Tissue'' (QIAGEN). Sterile deionized water was used to elute the DNA. The amplification of DNA was carried out on selected regions of four genes. In particular the barcoding region of the COI gene, and portions within ND5, EF-1α and PER genes were amplified. COI gene was selected as mainstream component of the analysis, and conversely ND5, EF-1α and PER genes were selected because only a little information is available on these DNA portions. A list of the used primers and their specifications are reported in Table 3. PCR was performed using 4 ml of the DNA extract as template for a 40 ml reaction final volume, using 0.5 ml of GoTaq R Flexi DNA Polymerase (Promega, Madison, WI, USA) per reaction. Each 40 ml reaction consisted of 8 ml of 5X Colorless GoTaq R Flexi Buffer (Promega), 4 ml of MgCl2 (25 mM), 1 ml of each of the two primers (10 pmol/ml), 1 ml of 10 mM nucleotide mix (Promega), and 20.5 ml sterile distilled water. Thermal cycler program used for the amplification consisted of an initial denaturation step at 95 ◦C for 1 min, followed by 35 cycles of 1 min at 95 ◦C, 1 min at the annealing temperature and 1 min at 72 ◦C; with a final